Modeling Subsurface Bioremediation by Geobacter

Geo bacter AcetateCarbon Dioxide U(VI) U(IV) e Uranium Contamination Removal Documented: Groundwaters from DOE Hanford Site Surface water from DOI site Washings from DOD contaminated soil

Acetate-Dependent Metal Reduction at Site 1103 U(VI) (µM) and NO 3 - (mM) % Fe (II)

Number of Geobacter sequences per gram of sediment Time (days) Nanograms of Geobacter 16S rDNA per gram of sediment micromolar U(VI) Percent Fe(II) Time (days) MPN and TaqMan results from site 1103 U(VI) and Fe(II) concentrations over time at site 1103

% of total clones recovered as Geobacteraceae

Acetate Solution (Low Conc.) DO < 1mg/L N2N2 Injection Gallery Pump or Gravity feed Acetate Groundwater Flow CH 3 COO - CO 2 Fe(III)Fe(II) CH 3 COO - U(VI)U(IV) U(VI) Zone of U(VI) Removal }

Subsurface Environments in Which Geobacteraceae Predominate 1. Fe(III) reduction zone of petroleum-contaminated aquifers 2. Uranium-contaminated subsurface sediments in which metal reduction was artificially stimulated 3. Field studies in which subsurface metal reduction was artificially stimulated 4. Fe(III) reduction zone of landfill-leachate contaminated aquifers 5. Diversity of Fe(III)-reducing aquatic sediments 6. On energy-harvesting electrodes in sediments Geographic Range: Minnesota, Mississippi, Wisconsin, Massachusetts, New Mexico, Canada, Switzerland, Netherlands Note: No Shewanella detected even with Shewanella-specific PCR primers

R load cm Fe(III)-Reducing Microorganisms Can Use Electrodes as an Electron Acceptor Harvesting Power From Aquatic Sediments and Other Sources of Waste Organics anode 8e - C2H4O2C2H4O2 2 CO 2 4H 2 O 8H+ Anode Reaction: C 2 H 4 O 2 + 2H 2 O  2 CO 2 +8H + + 8e - Cathode Reaction: 2O 2 +8H + + 8e -  4H 2 O water sediment cathode 2O 2 Sediment Battery e Bond, Holmes, Tender, and Lovley Science 295:

Who is enriched on the “anaerobic” (anode) electrode? % of clone library Over 75% of Anode population Delta-proteobacteria, primarily Geobacteraceae

Environmental Genomics with Geobacter Geobacter provides a rare instance in Environmental Microbiology in which it is possible to study organisms in pure culture that are closely related to the microorganisms that are known to be responsible for an process of interest in the environment. The study of Geobacter physiology is likely not only to indicate how these organisms reduce metals and electrodes in the subsurface, but also to elucidate other physiological factors which make microorganisms effective competitors in subsurface environments.

Elucidation of the Mechanisms for Electron Transfer in G. sulfurreducens Closely related to Geobacters that predominate in various environments Genome of G. sulfurreducens available Genetic system has been developed Methods for mass culturing available Techniques available for anaerobic biochemistry Can readily be grown in chemostats to provide physiologically consistent cells

GS AF PA AA r 2 = 0.70 p< 0.01 Data Provided by Barbara Methe, TIGR

Geobacter sulfurreducens cytochrome expression Fumarate Fe(III)

Cytochrome Expression Patterns

FerAFerBOrf4Orf2Orf3Orf1 Genes Organization of a known duplication region of G. sulfurreducens genome The ferA gene, encoded an outer membrane 89kD c-type cytochrome shares 79% identical sequences with the ferB gene Gene duplication: Two open reading frames-Orf1 and Orf2 preceded the ferB gene have 99.95% same sequences as those preceded ferA respectively.

Fe(III) Reduction by Mutants in the 89 kDa Cytochrome (ferA) and Its Homologue (ferB) Hours Fe(II) mM Wild type  ferA:: kan  ferB::cam

Soluble Fe(III) Fe(III)OOH in Mn(IV)OOH in Bar = 1µm Specific production of flagella by Geobacter when grown on insoluble substrates Childers, Ciufo, and Lovley Nature (in press)

1 µm Pili production in Geobacter

Fe(III) oxide reduction of pilA mutant

Reduction of soluble Fe(III) by  pilA

Figure 4 Chemotaxis assays using motile G. metallireducens. Plugs contained a, chemotaxis buffer; b,10 mM MnCl; c, Mn(IV) oxides; d, 10 mM FeSO 4 ; e, 50 mM Fe(III) oxides; f,10 mM Fe(III) oxides mM AQDS. Arrows emphasize bright ring of cells around agarose plugs.

NO 3 Motility is Not Needed to Access Soluble Electron Acceptors Fe(III) Oxide Motility is Needed to Find and Access Insoluble Electron Acceptors NO 3

Construction of a BAC library from sediment Collect sediment Extract DNA Electrophorese on low melting point agarose BAC Cloning Vector Excise DNA in agarose plugs Partial SgrA1 digest (Yields kb Fragments) Clone fragments ATCGATCAGCTCAGC GCATCAGCAGCTACG TAGCATCAGCATAAT GCATCGACGATCAGC GCATACGTAGCATCG Sequence Screen Library

1 10,000 20,000 20,001 30,000 40,000 40,001 50,000 60,000 Carbon metabolism rRNA Hypothetical Unknown Cell division Membrane Nucleotide Translation 16sTCA cycle FtsZTCA cycle tRNA synthase glucogenesis Desulfuromonas BAC clone from Uranium Bioremediation of Shiprock UMTRA Site

Future Directions More biochemical and genetic evaluation of potential electron carriers involved in electron transfer to metals and electrodes Begin evaluation of intermediary metabolism, stress response, and growth under nutrient-limiting conditions found in subsurface Begin evaluation of regulatory mechanisms Functional Genomics a. Proteomics (Carol Giometti, Argonne National Laboratory) b. Expression Analysis with DNA-Microarrays (Barbara Methe, TIGR) c. Genetic Studies

In Silico Biology (Bernhard Palsson) Development of a computer model of Geobacter functioning Comparative Environmental Genomics Genomic comparison of three genomes of pure cultures (G. sulfurreducens, G. metallireducens, D. acetoxidans) with Geobacter genomes recovered from subsurface environments Application of results from studies outlined above to measuring and predicting the activity of Geobacter in the subsurface Future Directions (continued)